It is known that many cell-surface proteins occur also in soluble forms. Some of these soluble molecules are created as such in vivo from distinct mRNAs, transcribed by alternative splicing mechanisms from the same genes which encode the cell-surface proteins. Others are derived from the cell-surface proteins presumably by proteolytic cleavage or, in the case of lipid anchored proteins, by the cleavage of their lipid anchor. Shedding of cell-surface proteins may occur spontaneously and, for example cancer cells seem to have a propensity for doing so. Shedding may also be induced by various stimulating agents.
Knowledge of the mechanisms involved in the shedding of cell-surface proteins and in its regulation is quite limited. None of the proteases or lipases taking part in it have so far been identified. There is also no clear indication of the subcellular site at which it occurs--on the cell surface or within some other intracellular compartment such as the lisozomes to which the cell-surface proteins are transported.
We have now been able to shed light on the mechanisms by which a cell surface protein which serves as a receptor for a cytokine, the p55 TNF receptor, is shed by cells. There are two distinct receptors, the p55 and p75 receptors, by which TNF, a cytokine produced primarily by mononuclear phagocytes, initiates its multiple effects on cell function. Both receptors are expressed in many cell types yet in differing amounts and proportions. The variation in their amounts seems to affect significantly the nature and intensity of the cellular response to TNF. One of the ways by which their expression is regulated is through induced shedding of the receptors. They can be shed in response to different kinds of inducing agents, depending on the type of cells. Granulocytes, for example, shed both receptors in response to the chemotactic peptide--fMLP (formylmethionylleucylphenyl-alanine) and shed specifically their p75 receptor when treated by TNF, while in T lymphocytes shedding of the p75 receptor, which is the predominant TNF receptor species in these cells, occurs upon antigen stimulation.
Shedding of both receptors may also effectively be induced by PMA (phorbol myristate acetate), by the serine phosphate inhibitor okadeic acid and by the calcium ionophore-A23187. The effect of PMA could be shown to reflect activation of protein kinase C, while the effect of okadeic acid seemed to involve the function of some other serine kinase. The amino acid sequences of the soluble forms of the two receptors which had been isolated from urine, correspond to sequences of a cysteine-rich module which extends along a major part of the extracellular domain of the two cell surface receptors. The C terminus of the urine-derived soluble form (Nophar Y., et al., EMBO J., Vol. 9, No.10, pp. 3269-3278 (1990)) of the p55 receptors was initially defined as Asn 172 which is located 11 residues upstream to the transmembranal domain of this receptor, while the C terminus of the soluble form of the p75 receptor corresponds to the residue located 44 amino acid upstream to the transmembranal domain of this receptor. However it was later revealed, that in urine also a somewhat longer soluble form of the p55 receptor, extending two further amino acids downstream towards the intracellular domain exists (Wallach D., et al., Tumor Necrosis Factor III, (Eds. T. Osawa and B. Bonavida) S. Karger Verlag (Basel) pp47-57 (1991). Whether these C termini correspond to the sites at which the receptor had initially been shed upon its release from the cell surface, or reflects also some further cleavage of the soluble form, occurring in the serum or the urine, is yet unknown.
Besides the impact of the shedding of the TNF receptors on the amounts of the cell-surface expressed receptors, this process also seems to contribute to the control of TNF function through effects of the soluble forms of the receptors, which maintain the ability to bind TNF and in doing so can affect its function in two, practically opposing, manners. On the one hand they inhibit the function of TNF by competing for it with the cell-surface receptors butt on the other hand, have also a stabilizing effect on TNF and can thus prolong its effects. The soluble forms of both species of the TNF receptor occur in human serum at concentrations which are normally very low, yet increase dramatically in various disease states, apparently due to enhanced receptor shedding, reaching levels at which they can effectively modulate TNF function.
To gain knowledge of the mechanisms of shedding of the TNF receptors we are attempting to identify the structural elements within the receptors which are involved in their cleavage. Previously we examined the effect of cytoplasmic deletions on the function and shedding of the pSS-TNF-R. We found that the signaling activity of the receptor depends on some function(s) of the C terminal part of the intracellular domain. However its shedding and the enhancement of the shedding by PMA occurs even in the complete absence of this domain (Brakebusch C., et al., EMBO J., Vol 11, pp. 943-950 (1992)).